Environmental Epigenetics, 2016, 1–9
doi: 10.1093/eep/dvv010
Review article
REVIEW ARTICLE
Medaka as a model for studying environmentally
induced epigenetic transgenerational inheritance
of phenotypes
Ramji K. Bhandari
Division of Biological Sciences, University of Missouri-Columbia, Columbia, MO 65211, USA
Correspondence address. Division of Biological Sciences, University of Missouri-Columbia, Columbia, MO 65211, USA. Tel: þ1-573-882-0983;
Fax: þ1-573-882-5020; E-mail [email protected]
Abstract
Ability of environmental stressors to induce transgenerational diseases has been experimentally demonstrated in plants,
worms, fish, and mammals, indicating that exposures affect not only human health but also fish and ecosystem health.
Small aquarium fish have been reliable model to study genetic and epigenetic basis of development and disease.
Additionally, fish can also provide better, economic opportunity to study transgenerational inheritance of adverse health
and epigenetic mechanisms. Molecular mechanisms underlying germ cell development in fish are comparable to those
in mammals and humans. This review will provide a short overview of long-term effects of environmental chemical
contaminant exposure in various models, associated epigenetic mechanisms, and a perspective on fish as model to study
environmentally induced transgenerational inheritance of altered phenotypes.
Key words: fish; environmental endocrine disrupting chemicals; transgenerational inheritance; epigenetics
Introduction
Environmental stress, a major contributor to evolution, enforces phenotypic variations in organisms that are directly exposed to it or remains as a causative factor for onset of
abnormal physical or behavioral health in offspring that had
never experienced it. This unique acquisition of phenotypic
traits primarily due to the ancestral experience to adverse situation is termed as transgenerational inheritance, and actual
mechanisms underlying such unprecedented environmental
health outcomes are not clear yet. Emergence of environmentally induced transgenerational phenotypes depends on nature
of stressor, window of development of the organism, and affected cell type. Because of organismal variability in reproductive processes and their response to environmental stress, a
wide variety of test models have been recommended to study
transgenerational inheritance. To date, the transgenerational
phenotypes, including those induced by chemical contaminants, have been shown in worms, flies, plants, fish, mice, rats,
domesticated farm animals, and humans, of which well-studied models are rodents and plants. Given that parental effects
are mediated to offspring via gametes, for a transgenerational
trait to develop, environmental stressors should affect germ
cells. Species that provide ample opportunities to study biology
of germ cells can serve as appropriate models to elucidate
mechanisms of transgenerational inheritance at the molecular
level. Each model has advantages over the other. Although rodents have been widely used, fish also have been found to be a
reliable, economic model to study molecular mechanisms underlying germ cell-mediated epigenetic transgenerational
Received 20 August 2015; revised 24 November 2015; accepted 8 December 2015
C The Author 2016. Published by Oxford University Press.
V
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inheritance of acquired traits. This review will briefly discuss
about current concepts on long-term effects of contaminants in
environmental ecotoxicology, evidences of long-term, transgenerational effects of endocrine disruptors (EDs), and the
medaka fish as a model to study environmentally induced epigenetic transgenerational inheritance.
Ecotoxicology and Long-Term Effects of
Contaminant Issues
During the mid-20th century, organophosphate chemicals were
being widely and ambitiously used to free humans from
unwanted bugs and pests. Rachel Carson, an environmentalist
and a marine biologist at the US Bureau of Fisheries, believed
that environmental problems in birds and aquatic wildlife were
caused by synthetic pesticides and warned the public of longterm effects of misusing them. She wrote in her 1962 book
“Silent Spring” [1, 2] asking readers to imagine “the world where
all animals and insects are dying, pollination does not occur,
greenery turns into brown, and no more birds sing the song for
you.” The book brought about a change in the US national policy
on pesticides, leading to a national ban on DDT and certain
other pesticides. Significant efforts have been made since then
to address issues in environmental toxicology at all levels.
Many comprehensive surveys have been conducted to measure
chemical contamination of aquatic resources and human exposures. Many toxic substances have been identified; and their
presence in the environment and organisms has been linked to
biological processes [3–7]. Many of the chemicals are “-cides,”
such as herbicides, pesticides, and rodenticides, used to remove
unwanted plants, insects, or rodents, respectively. Many others
belong to chemicals of everyday household, emergency, or personal use, such as active components of apparels, drugs, cosmetics, firefighting, and food packaging. Majority of these
chemicals are ubiquitous in nature and have often been found
to interfere with the synthesis, secretion, transport, binding, action, or elimination of natural hormones in the body that are responsible for the maintenance of homeostasis, reproduction,
development, and/or behavior [8–11]. Because of their ability to
disrupt endogenous hormone function, they are called as ED or
endocrine disrupting chemicals (EDCs).
Current risk assessment protocols are yet to include examination of long-term, multigenerational, and transgenerational
effects of EDCs on human and wildlife health. In many instances, there have been situations that the EDCs were widely
used for quite some time, remained in the market, and phased
out by the time, or even before, the long-term effects of these
chemicals began to appear in organisms. Currently, determining the effective concentration of chemicals that causes adverse
health effects in organisms has been a subject of active debate.
Highly variable results, mixed opinions from different scientific
communities (government, industry, and academia), and lack of
comprehensive risk assessment guidelines have restrained decision-making processes and led to the situation of dilemma
that eventually may pose humans and wildlife health at risk.
Direct and immediate toxic effects of exposure are the prime focus in risk assessment; however, emphasis should be given to
exposure effects that are initiated at the critical window of development and manifested as adverse health or behavioral outcomes later in life. Some chemicals that remain below the
current level of environmental relevance but surge a few folds
higher at certain times in a year can still cause adverse effects
when the surge/exposure occurs during the critical stage of life
history of the organism [9, 12]. These chemicals are often
termed as hit and run chemicals. It is, therefore, advisable to include in the current risk assessment guidelines the screening of
long-term, multigenerational and transgenerational effects of
chemical of concern using test models with shorter reproductive lifespan. Mechanistically, EDCs not only act as ligands for
selective receptors, or alter enzyme activities, but also induce
transcriptional activation or repression of genes via epigenetic
mechanisms. Differential gene expression is one of the criteria
for evaluating adverse outcome in exposed organisms.
Epigenetic changes precede transcriptional activation or repression of the target gene and serve as mechanistic links between
environment and physiology. Thus, inclusion of epigenetic
change as a component in adverse outcome pathway seems
necessary.
Environmentally Induced Adverse Health
Effects and Phenotypes
Inheritance of acquired traits, a theory postulated by Lamarck,
has been a topic of controversy for a long time and of utmost interest since experimental and epidemiological evidence suggested that certain environmentally induced phenotypic traits,
including acquired lifetime memory, can be transmitted to subsequent generations [12–19]. The traits that are acquired not as
a result of direct exposure to the stressor but due to the lifetime
experience of ancestors is called transgenerational traits.
Pioneering work of Dr Michael Skinner and co-workers laid the
foundation for research in environmental chemical-induced
transgenerational diseases [13]. Many of the chemicals tested so
far produced multiple disease or behavioral phenotypes at the
third or fourth generations [20–28]. To date, several forms of
transgenerational phenotypic traits, including those induced by
nutritional alterations and environmental chemical toxicants,
have been shown in worms [29, 30], flies [31], plants[32], fish [12,
33, 34], mice [14, 19, 35–37], domesticated farm animals [38, 39],
and humans [17, 40]. Mechanistic understanding of transgenerational inheritance of adverse health outcomes in model
species is gaining momentum, recently. On the other hand, evidence for transgenerational diseases in humans is derived from
epidemiological surveys [41], except for multigenerational and/
or transgenerational diseases in children and grandchildren of
diethylstilbestrol (DES)-prescribed women in the USA (CDC,
January 2012, http://www.cdc.gov/des/consumers/daughters/).
In the USA, an estimated 5–10 million persons were exposed to
DES during 1938–71, including women who were prescribed DES
while pregnant and the female and male children born of these
pregnancies. More than 30 years of research have confirmed
that health risks are associated with DES exposure. However,
not all exposed persons have developed health abnormalities.
Incidences of diseases such as clear cell carcinoma, reproductive tract deformities, pregnancy complications, and infertility
in DES Daughters and non-cancerous epididymal cysts, hypospadias, and infertility in DES sons have been reported to CDC.
Interestingly, animal studies mimicking human DES exposure
have shown disease phenotypes in mice similar to DES-exposed
humans, suggesting that results of the laboratory rodent exposure studies are translational to human health [42, 43].
Molecular studies as to whether DES induces transgenerational
diseases in the laboratory rodent models would be interesting
and valuable to define mechanisms underlying DES-induced
health effects in humans. Alternatively, fish can serve as excellent models to gain mechanistic insights into DES-induced phenotypic defects as fish have shown to develop reproductive
phenotypes in response to DES exposure [44].
Medaka model for transgenerational inheritance research
Diet, Transgenerational Effects, and Associated
Epigenetic Changes
“You are what you eat is a widely used proverb” but “you are
what your grandparents ate” will be an additional proverb in
the near future. Nutrition is one of the environmental factors
causing lasting transgenerational effects in a wide range of organisms, from worms to humans. For example, in a recent
study, nutritional restriction induced transgenerational longevity in worms, C. elegans [18]. The study found an epigenetic link
to transgenerational inheritance of phenotype. The small RNA
(22Gs) levels that follow L1-starvation-induced developmental
arrest were passed on for at least three generations. These small
RNAs targets included sets of genes involved in nutrition, such
as kin-29, gcy-23, polg-1, coq-3, and aco-1 [18]. Malnutrition during
gestation and perinatal life has been found to induce metabolic
syndromes and alteration in glucose metabolism in rats [45].
Another example of nutrition-induced transgenerational effects
is in humans. Epidemiological survey of different famines in the
Netherlands during World War II suggested transgenerational
effects of malnutrition on health and survival of grandchildren
[16, 17, 41]. Understanding the mechanisms underlying nutrition-induced transgenerational health problems and longevity
is currently an active area of research. Consumption of food
with phytoestrogens, compounds of plant origin with estrogenic
activity, has been linked to reproductive adverse outcomes in
ewes [46], cattle [47], and laboratory rodents [48]. In addition to
the action of phytoestrogens via estrogen receptors, peroxisome
proliferator-activated receptors, and the non-classical estrogen
receptor GPR30 [49], phytoestrogens have been found to alter
epigenetic marks by modulating activities of DNA and histone
methyltransferases, NAD-dependent histone deacetylases, and
other modifiers of chromatin structure [49–51]. Phytoestrogen
(cumestrol and equol) consumption increased DNA methylation
levels on the promoter regions of H-ras, a protooncogene, causing its silencing in neonatal mice [52], whereas genistein exposure caused activation of tumor suppressor genes in the mouse
prostate by modulating histone modifiers, such as histone
demethylation or acetylation [53]. Phytoestrogens affect reproduction and behavior in fish. In medaka (Oryzias latipes), reproductive problems caused by phytoestrogens include delayed
oocyte maturation in female at 0.75 and 30 mg genistein per fish
[54, 55], increased egg mortality and larval deformation of
brown trout (Salmo trutta) at 10 and 20 mg /l mixed phytosterols
found in pulp mill effluent [56], and impaired sexual differentiation in mosquitofish [57]. In humans, epidemiological data
show a strong association between incidences of reproductive
abnormalities and consumption of diet containing phytoestrogens [58, 59]. Despite several individual and species-specific variations in response to exposure to the nature of food, it is
concerning that the food has ability to change organism’s destiny by modulating non-genetic codes at the molecular level.
The nature of phytoestrogen action that is more or less identical
to mechanisms of actions of other EDCs and reproductive phenotypes of phytoestrogen exposure in laboratory models suggest that some transgenerational effects could be expected [59],
although strong evidence for such effects are still lacking.
Environmentally Induced Transgenerational
Inheritance and Epigenetic Mechanisms
Effectiveness of EDC exposure depends on affected cell type and
life history stages of the exposed organism. Exposure effects are
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profound during early development when tissues are still differentiating or during critical life history stages when tissues undergo age specific changes, such as puberty, lactation, and
aging. Effects on somatic cells, called somatic effects, are manifested as physiological responses and diseases in the exposed
individuals, whereas effects on germ cells can be transmitted to
subsequent generations via germ line (sperm or eggs) via epigenetic mechanisms. Understanding epigenetics is essential
for studying mechanisms underlying cell fate specification, tissue-specific gene expression, developmental origins of adult
disease, and transgenerational inheritance of disease. Most
extensively studied epigenetic processes are DNA methylation,
histone modifications, and small RNAs.
DNA Methylation
Epigenetic alterations, especially DNA methylation and histone
modifications, can be mitotically and meiotically stable [60],
therefore changes can potentially survive in a cell throughout
life. The reprogramming of DNA methylation initially occurs
upon fertilization in the zygote and secondly occurs in primordial germ cells (PGCs), which are the direct progenitors of sperm
or oocytes [61, 62]. It has, therefore, been believed that abnormal
epigenetic modifications in early life are detrimental to an organism’s later life health conditions. Any abnormal epigenetic
modifications can cause deregulation of the target gene [33].
Such modifications can have deleterious effects when they occur in stem cells or precursor cells since all differentiated cells
may carry abnormally programmed epigenome, thereby causing altered patterns of gene expression and molecular interactions in gene networks, consequently leading to an onset of
disease states.
Advent of next-generation sequencing has led to a significant advancement in understanding of biological processes. A
whole-genome bisulfite sequencing of cells undergoing reprograming, particularly during pre-implantation development in
mice, revealed some erasure-resistant genomic regions. These
protected genomic regions were intracisternal A particles, long
terminal repeats of endogenous retrovirus 1 (LTR-ERV1) elements, and a few single-copy sequences [63]. They may play significant roles in transmission of parental traits to the offspring.
Using a similar approach in mice, epigenetic reprogramming of
germ line cells has been studied. It has been found that a global
erasure of DNA methylation marks gives rise to a stem cell state
for PGCs, and de novo methylation starts allowing a controlled
gene expression pattern in germ cells in a gender specific manner [64]. A small portion (1%) of the PGC genome has been
found to be resistant to global DNA demethylation [65–68], suggesting that these genomic regions might be responsible for inheritance of parental phenotype. In rodent PGCs, DNA
demethylation continues 3 days past sex determination and
then de novo methylation occurs after day 16, coinciding with
mitotic arrest of spermatogonia in the developing testis and the
start of meiosis in ovaries. This window of germ cell reprogramming has been found to be susceptible to environmental chemical insult [69, 70].
So far, all DNA methylation marks identified in organisms
with environmentally induced transgenerational disease traits
are correlative; no causative links has been identified yet.
Hypotheses are that (i) environmental chemicals interfere with
DNA demethylation process in the PGCs during reprogramming
or (ii) they establish permanent methylation marks during de
novo methylation process. Therefore, differentiated germ cells
that develop into eggs or sperm also maintain these
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differentially methylated regions and these differentially methylated regions are passed on to the next generation via germ
line transmission without modification behaving as erasure-resistant imprint-like signatures. The individual with altered
germ cell epigenome may exhibit reproductive disease states
later in life, whereas the offspring originated by those germ cells
may exhibit disease states in both somatic cells and germ cells.
Epigenetic alterations, often termed as epimutations, may be
detectable in every cell of the offspring. However, yet, neither
an exposure-specific nor a phenotype-specific biomarker
(epimutations) has been identified to reliably predict exposure
history and transgenerational abnormalities. A recent controversial study by Iqbal et al. [71] has refuted the dogma that DNA
methylation marks are transmitted to subsequent generation
via germ line transmission. Mice that received previously
shown concentration of environmental contaminants to induce
transgenerational phenotypes did not establish the stable DNA
methylation marks in germ cells were supposed to be inherited
by the offspring germ cells. Although the study was well
designed, authors failed to show exposure-induced transgenerational phenotypes at F2 or F3 generations. Absence of phenotype somewhat supported the finding that no stable DNA
methylation marks were established in germ cells that could
be transmitted to subsequent generation. Moreover, a metaanalysis of diverse data sets related to ED-induced transgenerational gene expression alterations, including the data provided
by Iqbal et al. [71], suggested that effects of EDCs persist in unexposed generations [72].
Histone Modification
Histone modifications are major carriers of epigenetic information that both reflect and affect the transcriptional states of
underlying genes [73]. They include acetylation, phosphorylation, methylation, ubiquitination, and crotonylation [74].
Among histone modifications, histone lysine methylation appears to be the most stable. Among the different histone lysine
methylation states, H3K9 and H3K27 methylation appear to be
the most likely key regulators of classic epigenetic phenomena
[73]. For decades, it was thought that sperm histones are
replaced by protamines, and egg histone marks are reset after
fertilization and reestablished during preimplantation [75].
Recent studies suggest that histone modifications can be retained in the sperm [76–78] and inherited by offspring [79] and
are involved in epigenetic transgenerational inheritance of acquired traits [80, 81]. Hammoud et al. [76] unveiled the histone
marks that are retained in human sperm. H3K4me2 is enriched
at certain developmental promoters, whereas H3K4me3 is localized to a subset of developmental promoters, regions of HOX
gene clusters, certain non-coding RNAs, and paternally expressed imprinted loci. H3K27me3 is significantly enriched at
developmental promoters that are repressed in early embryo. In
C. elegans, multiple chromatin-modifying factors, including
H3K4me1/me2 and H3K9me3 methyltransferases, an H3K9me3
demethylase, and an H3K9me reader have been identified,
which either suppress or accelerate the progressive transgenerational phenotypes of spr-5 mutant worms [81]. Recently,
Siklenka et al. [80] showed that disruption of histone modifications, in the apparent absence of any changes in DNA methylation patterns, can also form the basis for transgenerational
transmission of epigenetic programming defects that can modify phenotypic characteristics in subsequent generations
[80, 82]. By overexpressing the human KDM1A histone lysine 4
demethylase
during
mouse
spermatogenesis,
authors
generated a mouse model producing spermatozoa with reduced
H3K4me2 within the CpG islands of genes implicated in development and studied the development and fitness of the offspring. KDM1A overexpression in one generation severely
impaired development and survivability of offspring. These defects lasted for two subsequent generations in the absence of
KDM1A germline expression. No apparent DNA methylation differences were observed in the CpG dense regions and changes
in expression and the phenotypic abnormalities observed in offspring correlated with altered histone methylation levels at
genes in sperm. These studies provide an evidence for complexity of transgenerational epigenetic inheritance involving multiple molecular factors, including the establishment of chromatin
states in spermatogenesis and sperm-borne RNA [80].
Small Non-Coding RNAs
Small non-coding RNAs are potential mediators of gene environmental interactions that can relay signals from environment
to the genome and exert regulatory function on gene activity
and dysregulation of genes in many diseases [83, 84]. The role of
sperm micro RNAs (miRNAs) in transmission of paternal experience and adverse offspring behavior has been emphasized recently. A study by Rodgers et al. [85] has found nine specific
miRNAs (miR-29c, miR-30a, miR-30c, miR-32, miR-193-5p, miR204, miR-375, miR-5323p, and miR-698) in the sire’s sperm sensitive to paternal stress. By injecting these miRNAs into the single cell stage embryo, the authors examined offspring level of
stress and associated molecular changes in paraventricular nucleus of brain using RNAseq. Study found that the paternal
sperm miRNAs function to reduce maternal mRNA stores in
early zygotes, ultimately reprogramming gene expression in the
offspring hypothalamus and recapitulating the offspring stress
dysregulation phenotype [86]. This study provides a mechanistic understanding of the transgenerational transmission of paternal lifetime experiences and offer valuable insight into the
novel factors influencing offspring disease risk and resilience.
In another study, early traumatic stress in early life was found
to alter miRNA expression in the sperm (miR-375-3p, miR-375-5p,
miR-200b-3p, miR-672-5p, and miR-466-5p) and behavioral and
metabolic responses in the progeny. Injection of these sperm
RNAs from traumatized males into fertilized wild-type oocytes
reproduced the behavioral and metabolic alterations in the resulting offspring, suggesting that ancestrally acquired traumatic
memory can be transgenerationally transmitted and miRNAs
play significant roles in mediating inheritance of environmentally induced traumatic stress to offspring [84]. Small RNA-induced gene silencing can persist over several generations via
transgenerationally inherited small RNA molecules in C. elegans
[29, 30]. In an elegant study with C. elegans, small RNAs seemed
to memorize lifetime experiences that were inherited by subsequent generations [18]. The study found that starvation-induced
developmental arrest, a natural and drastic environmental
change, leads to the generation of small RNAs that are inherited
through at least three consecutive generations. These small, endogenous, transgenerationally transmitted RNAs target genes
with roles in nutrition. Additionally, the F3 offspring of starved
animals showed an increased lifespan, corroborating the notion
of a transgenerational memory of past conditions. It will be
interesting to study how these miRNAs are generated by environmental influence, their dynamics during epigenetic reprogramming, and their circulating status to serve as biomarkers
of ancestral exposure. Together, transgenerational inheritance
is a buffered process that involves coordination of several
Medaka model for transgenerational inheritance research
epigenetic processes (DNA methylation, histone modifications,
and miRNAs) and transport of epigenetic hallmarks (epigenetic
memory) across generations finally leading to transcriptional
activation or silencing of target genes.
Medaka Fish (Oryzias latipes, Hd-rR Strain):
A Model to Study Environmentally Induced
Transgenerational Inheritance of Altered
Phenotypes
Studies from model organisms have significant translational
values as human subjects, or non-model wild species of concern, cannot be directly used to study these mechanisms.
Obviously, environmental chemicals that are capable of producing a phenotype in the model organisms should produce
similar effects in humans and domestic or wild animals but to
varying degrees given that the genetic make-up, body size,
food habits, and life history are different. Human exposure
data predominantly represent epidemiological survey-derived
correlative incidences of health and disease. However,
information on health of descendants of DES-prescribed pregnant women in the mid-20th century and phenotypes in laboratory rodent models with similar exposure history suggest
that mechanism of action of EDCs is conserved across taxa
[42, 43].
In the recent years, fish models, especially zebrafish, medaka, and fathead minnow, have been increasingly popular for
use in biomedical, ecological risk assessment, and environmental toxicology research. Genome sequences of zebrafish and medaka have been annotated, whereas that of fathead minnow is
in progress. Characterization of epigenetic processes has been
advanced in zebrafish and medaka mainly because of cutting
edge molecular tools, such as Crisper/Cas system, available for
gene manipulation, specific peptides and antibodies, and relevance to human health and disease [87–91]. High-throughput
sequencing of zebrafish germ cell epigenome (DNA methylation) has identified that epigenetic germ cell programming
events between mice and zebrafish are common [92, 93]. The
role of histones in regulation of key developmental genes has
been described in medaka [91] and studies underlying epigenetic reprogramming of cells during early development and
germ cell sex determination in medaka are currently in progress
(Bhandari et al., unpublished). Both medaka and zebrafish are
equally useful model organisms. Each model has advantages
over the other. Use of medaka (Oryzias latipes), which is also biomedical research model [94–98], may overcome the technical
limitations in understanding germ-cell-mediated transgenerational effects in mammals. Medaka sex determination occurs
between days 5 and 8 after fertilization. Germ cell reprogramming events are believed to be complementary to mouse [99].
Medaka have advantages over mice, including external fertilization and embryo development, daily spawning, availability of
large numbers of eggs and sperm, and virtually unlimited number of embryos produced by the same two parents to assign to
different treatment groups, along with a short generation time
(2 months) and easy, low-cost culture [96, 100]. In fish, transgenerational effects are manifested at F2 generation as F0 embryo,
and its F1 germ cells are directly exposed to test chemicals.
Zebrafish, however, may not be suitable for the study that
focuses on epigenetic modifications in sex-specific germ cells
because of gonadal hermaphroditism in early embryonic development and undefined sex determination in germ cells at the
stage of interest.
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Fish models have been proven to be useful for studying longterm, transgenerational effects of environmental stressors [12,
33, 34] and proposed as an alternative to mice especially with
regards to ease of their use, husbandry, economy, and resource
availability [33, 101]. Some species specificity of response to
chemicals exists. Issues such as life history, route of exposure,
rate of metabolism of chemical contaminants, mechanisms of
absorption and excretion, and genetic make-up are important
points to consider. However, the common mode of action and
conserved mechanism of cellular processes across taxa allow
scientists to interpret cross-species data from exposure studies.
In all species examined so far, the period of early embryonic development or the stage in the life history when further differentiation of tissues occurs has been found to be a susceptible
window to environmental stressors. For example, exposure of
medaka embryos to 100 mg/l bisphenol A (BPA) or 0.05 mg/l ethinylestradiol (EE2) only during the first 7 days of embryonic life
and never thereafter did not cause any gross phenotypic abnormalities in F0 and F1 generation adults, but the F2 and F3 adults
showed significant reduction in fertilization capacity and resultant F3 and F4 embryos showed a reduced rate of survival [12].
The transgenerational phenotype was induced just because of
exposure to these chemicals at the critical window of embryonic development. Although the concentrations used were
above the level of current environmental relevance but were
within the level of annual surge in the natural environment
[9, 102, 103], indicating that irrespective of exposure to environmental relevant levels of chemicals exposure to hit and run
concentration of chemicals at the critical window of development can cause a significant damage to a population via transgenerational effects. Characterization of epigenetic processes
(histone modifications, DNA methylation, and circulating
miRNA profiles) associated with transgenerational inheritance
of altered phenotypes, these fish is currently in progress
(Bhandari et al., unpublished). In zebrafish, exposure to waterborne dioxin (50 pg/ml) at 3 weeks postfertilization (wpf) and
again at 7 wpf stage caused a reduction in egg release and fertilization success, a female skewed sex ratio, and skeletal kinks at
F1and F2 generation [33]. Paternal exposure to BPA during spermatogenesis increased rate of heart failures of progeny up to F2
generations in zebrafish [104]. Parental exposure of zebrafish to
a varying concentration of Benzo[A]Pyrene (BaP) for 21 days resulted in body morphology deformities (shape of body, tail, and
pectoral fins) that continued to F2 and F3 generations, whereas
craniofacial structures (length of brain regions, size of optic and
otic vesicles, and jaw deformities), although not significantly affected in the F1 generation, emerged as significant deformities
in the F2 generation [34], suggesting human health risks of
exposure to BaP. In a live bearing fish guppy, Poecilia reticulate,
maternal exposure to 20 ng/l EE2 until birth caused a transgenerational anxiety phenotype in the F2 generation offspring
[105]. Studies showing occurrence of environmentally induced
transgenerational inheritance of phenotypes in fish are accumulating rapidly and similar phenotypes in other species are
anticipated. These increasing numbers of studies in fish and
other model species indicate that environmental EDCs can induce transgenerational effects relevant to human and ecosystem health. Although molecular mechanisms underlying
transgenerational inheritance of environmental phenotype in
these fish are not yet clarified, unraveling of associated genetic
and epigenetic changes is, however, expected.
Irrespective of the route of exposure, it is important to consider how much of the effective chemical was taken by the exposed organism. In a study with medaka, uptake concentration
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Environmental Epigenetics, 2016, Vol. 2, No. 1
of both BPA and EE2 was measured by using radiolabeled
chemicals (3H-BPA, 3H-EE2) [12]. Out of 100 mg/l 3H-BPA, medaka
embryo’s uptake was only 178 pg/mg of egg and 3H-EE2 was
4 pg/mg egg out of 0.05 mg/l. F2 and F3 generation fish from the
exposure lineage developed transgenerational abnormalities. It
is, therefore, possible that an exposure to very small amount of
chemical is likely sufficient to establish chemical signatures on
the genome (i.e. epigenome) that subsequently lead to adverse
health and disease in subsequent generations. Effects in cells
that proliferate slowly would take comparatively longer to appear than that in cells that proliferate rapidly. Some effects
seem to be persistent across multiple generations and some detectable only in the third or fourth generation. The investigation
on mechanisms mediating such transgenerational effects are
currently of the best interest to scientific community and government agencies.
5.
6.
7.
8.
9.
Conclusions
The basic molecular mechanisms of gene regulation are highly
conserved in all eukaryotic organisms. Diseases such as metabolic syndromes, neurological disorders, infertility, cancer, and
increased susceptibility to microbial diseases have developmental origins and epigenetic links. Given that the mode of action of environmental EDCs is common in organisms across
taxa, the downstream molecular pathways and gene networks
associated with development of disease and behavioral phenotypes, although highly variable and organism-specific, are likely
to share some common routes. Epigenetics links environment
with physiology. It seems crucial to find chemical-specific epigenetic signatures and the common downstream routes to phenotypes to develop the biomarkers that are reliably predictive of
exposure and disease phenotype. Fish can serve as economic,
easy, and convenient model to study environmentally induced
epigenetic transgenerational inheritance of phenotypes.
Acknowledgements
The authors thank undisclosed reviewers for suggestions and
critiques on the manuscript. This work was supported by a
grant from US Geological Survey, Cooperative Ecosystem
Studies Unit (G15AS00092) to R.K.B. Currently, R.K.B. is a visiting research scientist at U. S. Geological Survey, Columbia
Environmental Research Center. Any use of trade, firm, or
product names is for descriptive purposes only and does not
imply endorsement by the U.S. Government.
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